Field of the Invention
The present invention relates generally to chromatography, and more particularly to high performance liquid chromatography or supercritical fluid chromatography systems, and still more particularly to a system and method for improving the sensitivity and, hence, the accuracy and precision of these techniques. It should be noted that methodology that is commonly referred to as supercritical fluid chromatography is, in some cases, done under conditions that are, in fact, subcritical. Therefore, it should be clarified that all references to supercritical fluid chromatography, in this patent application, are meant to also encompass subcritical fluid chromatography.
Background Discussion
Chromatographers often desire to obtain better sensitivity. A simple way to accomplish this is to increase the injection volume. However, the analyst has only a limited ability to do this as the chromatography will begin to degrade when larger volumes are injected. This is due to two phenomena: First, there is a distortion or “smearing” effect that occurs as the injection solvent (or diluent) begins to mix with the mobile phase solvent. This occurs because when two different solvents begin to mix (even when the solvents are completely miscible with one another) they do not dissolve into one another immediately. As a result, some distortion and spreading of the injected sample occurs. Second, as an increasingly larger volume is injected, there is some degree of band broadening due directly to the volume of the injection itself.
In some cases, the use of larger injection volumes may be possible simply by using an injection solvent that is chromatographically weaker than the mobile phase (predominantly aqueous in the case of reversed phase liquid chromatography). Assuming the method parameters are such as to enable efficient focusing, this would allow for larger injection volumes, despite the issues described above, inasmuch as the solute (or analyte) peaks would be focused into sharper bands at the head of the analytical column. However, this option is only available in situations where the solutes have sufficient solubility, and where the sample matrix is sufficiently dissolved or dispersed, in these chromatographically weak injection solvents. In the majority of cases this is not possible, and injection solvents chromatographically stronger than the mobile phase must be used. The use of these stronger injection solvents results in the mobile phase being transiently stronger during the time when the injected sample is being transferred onto the column and, therefore, very inefficient focusing occurs at the head of the column.
Therefore, other than in those limited cases where chromatographically weak injection solvents can be used, a different approach must be found to increase the injection volume. The approaches reported in the literature have generally made use of column switching techniques. These systems often use a two-part process: in the first step a large volume is injected and concentrated onto a trap, and in the second step a switching valve is opened or switched and the solutes are transferred from the trap onto the analytical column. This technique has also been used with a membrane, and a solid-phase-microextraction fiber as the trap. Because of the complex nature of these processes, these approaches can be harder to run and more difficult in terms of troubleshooting and training. In addition, these types of setups are undesirable in pharmaceutical GMP environments, as the additional hardware must be formally qualified. Finally, these approaches can be problematic in terms of the additional dead volume contributed by the extra hardware that is needed.